DESCRIPTION: Dr. Jeffrey Way requests four years of funding to continue his research on the genetic control of cell type in C. elegans. Dr. Way has been studying the mec-3 gene of C. elegans, which encodes a DNA- binding protein required for cell fate determination of the touch cell neurons. In C. elegans, as in other animals, cell type is determined by expression of specific regulatory genes that activate functionally related structural genes. To ask how cell type-specifying genes are regulated, he is studying the mec-3 gene. mec-3 is expressed at the end of a stereotyped cell lineage in only one of two daughter cells after a terminal cell division. These studies are relevant for understanding cell polarity and asymmetric cell division. The sequences in the mec-3 promoter that are necessary to establish mec-3 expression will be identified. Of particular interest is determining whether the unc-86 gene is sufficient for expression of mec-3. Sequences will be defined using a heterologous system for assaying mec-3 enhancers, synthetic oligonucleotides corresponding to candidate binding sites, and deletions and point mutations in the intact mec-3 promoter. Factors that bind to the minimal mec-3 establishment sequences will be sought either by probing expression libraries with the minimal sequence or by biological strategies (repression in E. coli, or transcriptional activation in yeast). Dr. Way will also be studying the unc-73 gene, which encodes the cdc-24 homolog in C. elegans, the C. elegans cdc-42 homolog and genes that interact with unc-73. cdc-42 is a small GTP-binding protein implicated in budding in yeast and cdc-24 is a guanine nucleotide exchange factor that appears to act on cdc-42. These molecules are conserved throughout eukaryotes and some evidence suggests that they are involved in setting up cell polarity in mammalian cells as well as yeast cells. The unc-73 gene in C. elegans is required for normal axon guidance, cell migration, and asymmetric cell division. Some oncogenes are related to cdc-24 in sequence and therefore it is hoped that understanding this group of genes may shed light on human tumorigenesis. The relationship between unc-73, the cdc-24 homolog, sup-73, a suppressor of unc-73, cdc-42, and asymmetric divisions that generate mec-3 expressing cells will be examined. It will be verified that unc-73 mutations actually cause a defect in asymmetric cell division. At this point it seems that sup-73 and cdc-42 are not the same gene, so null mutations in cdc-42 will be sought by transposon mutagenesis. The dosage of cdc-42 will be increased by introducing extra copies or heat shock to see if asymmetric cell division is altered. The interaction between unc-73 and sup-73 will be characterized further. More suppressors of unc-73 will be sought. Mosaic analysis of unc-73 function will be conducted to see where this gene acts. sup-73 will be cloned. Genes that interact with unc-73 will be sought. Double mutants will be made between genes that are implicated in asymmetric cell division. New mutations will be sought that are lethal in combination with unc-73. The vab-8 gene is implicated in asymmetric cell division, axon guidance, and cell migration. This gene will be cloned and characterized. The hypothesis that cdc-42 is involved in generating mother-daughter asymmetry that allows mating type switching to be regulated between mother and daughter cells in yeast will be tested. cdc-42 protein forms a cap in the bud tip and becomes localized to the daughter cell. cdc-42 will be overproduced to see whether expression of cdc-42 at high levels in the mother cell can inhibit expression of HO and mating type switching.